Although non-viral vectors are believed to hold high potential for future large scale clinical gene therapy applications, their use is currently limited by their yield, which is several orders of magnitude smaller than for the major viral vector systems. Multiple causes, in variable proportions depending on the specific formulation, are responsible for the overall low yield of non-viral vectors. In all cases, a factor reducing the yield by one to three orders of magnitude is the inefficient translocation of the DNA from the cytoplasm to its final destination, the nucleus. In most cells of the mature organism, which are postmitotic, the translocation is blocked by an intact nuclear membrane. Although attempts have been made to improve the nuclear translocation of the vectors by attaching to DNA synthetic peptides or proteins containing Nuclear Localization Signals (NLSs), improvements have been minor. The situation is expected to be even worse for future tentatively optimized situations, when very few copies of the vector will be present in each cell. The aim of this project is to explore a new principle, which holds the promise for much more efficient nuclear translocation. As the protein import pathway is based on a chain of interactions of the proteins to be imported with transport factors, the central idea of this project is to place the vector DNA not at the entry point of this chain, where it has to compete with the vast number of native proteins waiting to be imported, but at the end of the chain, right before the final step of crossing the nuclear pore. In biochemical terms this concept translates into using as targeting moiety not an NLS, but the transport factors karyopherins beta, which in normal situations dock the complexes at the nuclear pores. All three known human karyopherins beta will be investigated for their ability to act as nuclear targeting moieties. The smallest fragment of each karyopherin retaining full translocation capacity will be determined, for use in improved delivery systems. We expect that this approach will assure rapid and highly efficient translocation of the DNA across the nuclear membrane.